Characterization of metabolic activation of pentachlorophenol to quinones and semiquinones in rodent liver

Pentachlorophenol (PCP), a widely used biocide, induces liver tumors in mice but not in rats. Metabolic activation of PCP to chlorinated quinones and semiquinones in liver cytosol from Sprague-Dawley rats and B6C3F1 mice was investigated in vitro (1) with microsomes in the presence of either beta-nicotinamide adenine dinucleotide phosphate (NADPH) or cumene hydroperoxide (CHP), (2) with CHP in the absence of microsomes, and (3) with horseradish peroxidase (HRP) and H2O2. Mono-S- and multi-S-substituted adducts of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) and Cl4-1,2-BQ and their corresponding semiquinones [i.e. tetrachloro-1,4-benzosemiquinone (Cl4-1,4-SQ) and tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ)] were measured by gas chromatography-mass spectrometry (GC-MS). Qualitatively, the metabolites of PCP were the same in both rats and mice for all activation systems. Induction of PCP metabolism by either 3MC or PB-treated microsomes was observed in NADPH- but not in CHP-supported systems. In rats, the amount of induction was comparable with either 3MC or PB. 3MC was a stronger inducer than PB in mice and also induced a greater amount of metabolism than in rats. This suggests that induction of specific P450 isozymes may play a role in the toxicity of PCP to mice. Both HRP/H2O2 and CHP led to production of the full spectrum of chlorinated quinones and semiquinones, confirming the direct oxidation of PCP. CHP (with or without microsomes) converted PCP into much greater quantities of quinones and semiquinones than did microsomal P450/NADPH or HRP/H2O2 in both species. This implies that, under conditions of oxidative stress, endogenous lipid hydroperoxides may increase PCP metabolism sufficiently to enhance the toxicity and carcinogenicity of PCP.     

Naturally occurring quinols and quinones studied as semiquinones by electron spin resonance

A simple procedure using electron spin resonance techniques was applied to detect, identify and quantify quinones and quinols in crude plant extracts. Hydroquinone was determined in Pyrus, plumbagin in Drosera and Ceratostigma, and hydrojuglone in Juglandaceae. Hydrojuglone is found in markedly higher concentrations in Pterocarya and in Juglans than in Carya. Plastoquinol has been observed in 500 of 700 plant extracts studied. Esters of phenolic acids are easily detected and distinguished, e.g. chlorogenic and rosmarinic acids. Esters of homoprotocatechuic and of dihydrocaffeic acid occur widely in the Oleaceae. The limitations of the method are discussed.     

Ethyl pyruvate-mediated Nrf2 activation and hemeoxygenase 1 induction in astrocytes confer protective effects via autocrine and paracrine mechanisms

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule under various pathological conditions, such as, during cerebral ischemia and sepsis in animal models. Here, the authors investigated the novel molecular mechanism underlying the anti-oxidative effect of EP in primary astrocyte cultures, particularly with respect to nuclear factor E2-related factor 2 (Nrf2) activation and hemeoxygenase 1 (HO-1) induction. EP was found to induce Nrf2 translocation and the inductions of various genes downstream of Nrf2 and these resulted in the amelioration of the oxidative damage of H(2)O(2). Furthermore, EP dose-dependently suppressed H(2)O(2)-induced astrocyte cell death (12h preincubation with 5mM EP increased cell survival after 1h exposure to 100 μM H(2)O(2) from 32.6±0.7% to 63±1.8%). HO-1 was markedly induced (4.9-fold) in EP-treated primary astrocyte cultures and Nrf2 was found to translocate from the cytosol to the nucleus and bind to the antioxidant response element (ARE) located on HO-1 promoter after EP treatment. siRNA-mediated HO-1 or Nrf2 knockdown and zinc protoporphyrin (ZnPP)-mediated inhibition of HO-1 activity showed that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of EP, which was found to involve the ERK and Akt signaling pathways. Furthermore, EP-conditioned astrocyte culture media was found to have neuroprotective effects on primary neuronal cultures exposed to oxidative or excitotoxic stress, and this seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and glutathione (GSH), which accumulated in EP-treated astrocyte culture media. Interestingly, we also found that in addition to HO-1, EP-induced Nrf2 activation increased the expressions of various anti-oxidant genes, including GST, NQO1, and GCLM. The study shows that EP-mediated Nrf2 activation and HO-1 induction in astrocytes act via autocrine and paracrine mechanisms to confer protective effects.     

Hydrogen sulfide protects astrocytes against H(2)O(2)-induced neural injury via enhancing glutamate uptake

Excess extracellular glutamate, the main excitatory neurotransmitter, may result in excitotoxicity and neural injury. The present study was designed to study the effect of hydrogen sulfide (H(2)S), a novel neuromodulator, on hydrogen peroxide (H(2)O(2)) -induced glutamate uptake impairment and cellular injuries in primary cultured rat cortical astrocytes. We found that NaHS (an H(2)S donor, 0.1-1000 microM) reversed H(2)O(2)-induced cellular injury in a concentration-dependent manner. This effect was attenuated by L-trans-pyrrolidine-2,4-dicarboxylic (PDC), a specific glutamate uptake inhibitor. Moreover, NaHS significantly increased [(3)H]glutamate transport in astrocytes treated with H(2)O(2), suggesting that H(2)S may protect astrocytes via enhancing glutamate uptake function. NaHS also reversed H(2)O(2)-impaired glutathione (GSH) production. Blockade of glutamate uptake with PDC attenuated this effect, indicating that the effect of H(2)S on GSH production is secondary to the stimulation of glutamate uptake. In addition, it was also found that H(2)S may promote glutamate uptake activity via decreasing ROS generation, enhancing ATP production and suppressing ERK1/2 activation. In conclusion, our findings provide direct evidence that H(2)S has potential therapeutic value for oxidative stress-induced brain damage via a mechanism involving enhancing glutamate uptake function.     

Preconditioning by sesquiterpene lactone enhances H2O2-induced Nrf2/ARE activation

The Nrf2/ARE pathway plays a pivotal role in chemoprevention and neuroprotection. Here, we report that sesquiterpene lactones extracted from Calea urticifolia and feverfew increased enhancer activity of the ARE. ARE activation was dependent on the number of α,β-unsaturated carbonyl groups each compound bears and calealactone A (CL-A) harboring 3 of those was the most potent ARE inducer. At subtoxic doses, CL-A induced expression of heme oxygenase-1 (HO-1) gene, one of ARE target genes, through activation of the Nrf2/ARE pathway involving transient ROS generation and activation of PI3-K/Akt and MAPK pathways. Interestingly, H₂O₂-induced ARE activation and HO-1 induction were potentiated by pretreatment with CL-A at lower concentrations, at which Nrf2/ARE activation by the compound was minimal. These results suggest a possibility that preconditioning by sesquiterpene lactone may enhance activation of the Nrf2/ARE pathway and induction of phase II detoxification/antioxidant enzymes upon oxidative stress, thereby resulting in increased resistance to oxidative damage.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O(2) into superoxide radical, later transformed into H(2)O(2). This enzymatic activity requires previous activation with either 3 mM Mn(2+) or guanosine 5'-0-(3-thiotriphosphate) (GTPgammaS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S(0.5) for NADPH was 44 microM; S(0.5) for FAD was 8 microM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPgammaS plus adrenaline was dose- and Ca(2+)-dependent and proceeded through alpha(1)-adrenergic receptors (AR), whereas beta-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H(2)O(2) as additional transduction signal for adrenaline response in hepatic cells.     

Horse heart myoglobin catalyzes the H2O2-dependent oxidative dehalogenation of chlorophenols to DNA-binding radicals and quinones

The heme-containing respiratory protein, myoglobin (Mb), best known for oxygen storage, can exhibit peroxidase-like activity under conditions of oxidative stress. Under such circumstances, the initially formed ferric state can react with H2O2 (or other peroxides) to generate a long-lived ferryl [Fe(IV)=O] Compound II (Cpd II) heme intermediate that is capable of oxidizing a variety of biomolecules. In this study, the ability of Mb Cpd II to catalyze the oxidation of carcinogenic halophenols is demonstrated. Specifically, 2,4,6-trichlorophenol (TCP) is converted to 2,6-dichloro-1,4-benzoquinone in a H2O2-dependent process. The fact that Mb Cpd II is an active oxidant in halophenol dehalogenation is consistent with a traditional peroxidase order of addition of H2O2 followed by TCP. With 4-chlorophenol, a dimerized product is formed, consistent with a mechanism involving generation of a reactive phenoxy radical intermediate by an electron transfer process. The radical nature of this process may be physiologically relevant since recent studies have revealed that phenoxy radicals and electrophilic quinones, specifically of the type described herein, covalently bind to DNA [Dai, J., Sloat, A. L., Wright, M. W., and Manderville, R. A. (2005) Chem. Res. Toxicol. 18, 771-779]. Thus, the stability of Mb Cpd II and its ability to oxidize TCP may explain why such compounds are carcinogenic. Furthermore, the initial rate of dehalogenation catalyzed by Mb Cpd II is nearly comparable to that of the same reaction carried out by turnover of the ferric state, demonstrating the potential physiological danger of this long-lived, high-valent intermediate.     

Diverse catalytic activities in the alphabeta-hydrolase family of enzymes: activation of H2O, HCN, H2O2, and O2

The article describes the observation of novel catalytic activities in the alphabeta-hydrolase superfamily apparently unrelated to ester hydrolysis and unexpected biochemical observations relating to the structure and function of the serine catalytic triad in these enzymes. One common feature of these novel activities is the activation of a small diatomic molecule, but via diverse chemistry. Possible mechanisms of catalysis are discussed.     

Retinal astrocytes pretreated with NOD2 and TLR2 ligands activate uveitogenic T cells

On entering the tissues, infiltrating autoreactive T cells must be reactivated locally to gain pathogenic activity. We have previously reported that, when activated by Toll-like receptor 3 (TLR3) and TLR4 ligands, retinal astrocytes (RACs) are able to function as antigen-presenting cells to re-activate uveitogenic T cells and allow responder T cells to induce uveitis in mice. In the present study, we found that, although the triggering of TLR2 or nucleotide-binding oligomerization domain receptor 2 (NOD2) alone did not activate RACs, their combined triggering induced RACs with the phenotypes required to efficiently re-activate interphotoreceptor retinoid-binding protein (IRBP)-specific T cells. The synergistic effect of TLR2 and NOD2 ligands on RAC activation might be explained by the observations that bacterial lipoprotein (BLP, a TLR2 ligand) was able to upregulate NOD2 expression and the combination of BLP and muramyldipeptide (MDP, a NOD2 ligand) enhanced the expression of RICK (Rip2), the signaling molecule of NOD2. Moreover, the synergistic effect of MDP and BLP on RACs was lost when the RACs were derived from NOD2 knockout mice or were pre-treated with Rip2 antagonist. Thus, our data suggest that exogenous or endogenous molecules acting on both TLR2 and NOD2 on RACs might have an enhancing effect on susceptibility to autoimmune uveitis.     

Naringenin Attenuates H2O2-Induced Mitochondrial Dysfunction by an Nrf2-Dependent Mechanism in SH-SY5Y Cells

Neurochemical Research - Mitochondria are the major site of ATP production in mammalian cells. Furthermore, these organelles are a source and a target of reactive oxygen species (ROS), such as...     

Nrf2 activity is lost in the spinal cord and its astrocytes of aged mice

We report herein a study of aging using in vitro and in vivo models. Glial fibrillary acidic protein and ferritin expression levels increased, and the levels of glutamate transporter 1 and transferrin receptor 1 decreased in aging mouse spinal cord and its astrocytes. Mitochondrial transmembrane potential in astrocytes decreased after 60 d of culture. Given the relationship between aging and loss of antioxidant tolerance capacity, we examined the expression of heme oxygenase 1 (HO1) and NAD(P)H/quinone oxidoreductase 1 (NQO1) in the old mouse astrocytes and spinal cord. Indeed, both antioxidant enzymes decreased there. Total nuclear factor E2-related factor 2, which governs basal and inducible expression of HO1 and NQO1, decreased significantly. Significantly, epigallocatechin gallate restored the Nrf2 activity.     

Mitochondrial H2O2 regulates the angiogenic phenotype via PTEN oxidation

Recent studies have demonstrated that the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), the antagonist of the phosphosphoinositol-3-kinase (PI3K) signaling cascade, is susceptible to H2O2-dependent oxidative inactivation. This study describes the use of redox-engineered cell lines to identify PTEN as sensitive to oxidative inactivation by mitochondrial H2O2. Increases in the steady state production of mitochondrial derived H2O2, as a result of manganese superoxide dismutase (Sod2) overexpression, led to PTEN oxidation that was reversed by the coexpression of the H2O2-detoxifying enzyme catalase. The accumulation of an oxidized inactive fraction of PTEN favored the formation of phosphatidylinositol 3,4,5-triphosphate at the plasma membrane, resulting in increased activation of Akt and modulation of its downstream targets. PTEN oxidation in response to mitochondrial H2O2 enhanced PI3K signaling, leading to increased expression of the key regulator of angiogenesis, vascular endothelial growth factor. Overexpression of PTEN prevented the H2O2-dependent increase in vascular endothelial growth factor promoter activity and immunoreactive protein, whereas a mutant PTEN (G129R), lacking phosphatase activity, did not. Furthermore, mitochondrial generation of H2O2 by Sod2 promoted endothelial cell sprouting in a three-dimensional in vitro angiogenesis assay that was attenuated by catalase coexpression or the PI3K inhibitor LY2949002. Moreover, Sod2 overexpression resulted in increased in vivo blood vessel formation that was H2O2-dependent as assessed by the chicken chorioallantoic membrane assay. Our findings provide the first evidence for the involvement of mitochondrial H2O2 in regulating PTEN function and the angiogenic switch, indicating that Sod2 can serve as an alternative physiological source of the potent signaling molecule, H2O2.     

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.     

Saturated long-chain fatty acids activate inflammatory signaling in astrocytes

This study describes the effects of long-chain fatty acids on inflammatory signaling in cultured astrocytes. Data show that the saturated fatty acid palmitic acid, as well as lauric acid and stearic acid, trigger the release of TNFα and IL-6 from astrocytes. Unsaturated fatty acids were unable to induce cytokine release from cultured astrocytes. Furthermore, the effects of palmitic acid on cytokine release require Toll-like receptor 4 rather than CD36 or Toll-like receptor 2, and do not depend on palmitic acid metabolism to palmitoyl-CoA. Inhibitor studies revealed that pharmacologic inhibition of p38 or p42/44 MAPK pathways prevents the pro-inflammatory effects of palmitic acid, whereas JNK and PI3K inhibition does not affect cytokine release. Depletion of microglia from primary astrocyte cultures using the lysosomotropic agent l-leucine methyl ester revealed that the ability of palmitic acid to trigger cytokine release is not dependent on the presence of microglia. Finally, data show that the essential ω-3 fatty acid docosahexaenoic acid acts in a dose-dependent manner to prevent the actions of palmitic acid on inflammatory signaling in astrocytes. Collectively, these data demonstrate the ability of saturated fatty acids to induce astrocyte inflammation in vitro. These data thus raise the possibility that high levels of circulating saturated fatty acids could cause reactive gliosis and brain inflammation in vivo, and could potentially participate in the reported adverse neurologic consequences of obesity and metabolic syndrome.